PCR reaction vessel, PCR device, and PCR method

ABSTRACT

A PCR reaction vessel includes: a substrate; a channel formed on the substrate; a pair of filters, a first filter and a second filter, provided at respective ends of the channel; a pair of air communication ports, a first air communication port and a second air communication port, that communicate with the channel through the first filter and the second filter; a thermal cycle region formed between the first filter and the second filter in the channel; a branch point formed between the first filter and the second filter in the channel; a branched channel whose one end is connected to the branch point; and a sample introduction port formed at the other end of the branched channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional of application Ser. No. 15/993,814 filed May 31,2018, now issued as U.S. Pat. No. 10,988,800 on Apr. 27, 2021, claimingpriority based on International Application No. PCT/JP2016/085216 filedNov. 28, 2016, claiming priority based on Japanese Patent ApplicationNo. 2015-235129 filed Dec. 1, 2015, the disclosures of which areincorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to PCR (Polymerase Chain Reaction)reaction vessels used for polymerase chain reactions and to PCR devicesand PCR methods in which the PCR reaction vessels are used.

BACKGROUND ART

Genetic testing is widely used for examinations in a wide variety ofmedical fields, identification of farm products and pathogenicmicroorganisms safety assessment for food products, and even forexaminations for pathogenic viruses and a variety of infectiousdiseases. In order to detect with high sensitivity a minute amount ofgene's DNA, methods of analyzing the resultant obtained by amplifying aportion of DNA are known. Above all, a PCR method is a remarkabletechnology where a certain portion of a very small amount of DNAcollected from an organism or the like is selectively amplified. In aPCR method, a predetermined thermal cycle is applied to a sample inwhich a biological sample containing DNA and a PCR reagent consisting ofprimers, enzymes, and the like are mixed so as to cause reactions suchas denaturation, annealing, and elongation to be repeated so that aspecific portion of DNA is selectively amplified.

It is a common practice to perform a PCR method by putting apredetermined amount of a target sample into a PCR tube or a reactionvessel such as a microplate (microwell) in which a plurality of holesare formed. However, in recent years, it has been put to practical useto perform a PCR method while using a reaction vessel (also referred toas “chip”) provided with a micro-channel that is formed on a substrate.In any reaction vessel, the progress of various technologies allow apredetermined thermal cycle to be provided with high speed and highaccuracy in the reaction vessel.

Patent document No. 1 discloses a reaction vessel in which a channel forperforming PCR is formed. In this reaction vessel, the channel is formedbetween two overlapping resin substrates, and a sample introduction portfor introducing a sample into the channel and a sample discharge portfor discharging the sample via through holes formed in the resinsubstrates are ensured. In a recessed portion on the back surface of aresin substrate, a temperature adjusting unit consisting of, forexample, a Peltier element or the like is arranged. A nozzle is arrangedat the sample introduction port of the reaction vessel, and the samplecan be moved in the channel by feeding and/or suctioning the air throughthe nozzle.

-   [Patent document No. 1] Japanese Patent Application Publication No.    2009-232700.

SUMMARY OF THE INVENTION

In a PCR method, it is necessary to prevent contamination into thesystem from the outside during the processing of a sample. If acontamination includes biological pieces or the like other than those ofan object to be processed, there is a possibility of amplifying DNAcontained in the biological pieces other than those of the object to beprocessed. In this case, the analysis that follows cannot be accuratelyperformed using the sample to be processed. Therefore, after introducingthe sample into the reaction vessel, it is necessary to take measures inorder to prevent contamination.

However, in the invention disclosed in Patent document No. 1, forexample, if biological pieces other than those of the object to beprocessed are attached to the nozzle or a pump that sends the air to thenozzle, the biological pieces other than those of the object to beprocessed may enter the reaction vessel through the sample introductionport, and contamination may thus occur. Also, it is not realistic fromthe cost and environmental aspects to discard nozzles, tip parts ofpumps, attachments, and the like every single time a PCR process isperformed.

In this background, a purpose of the present invention is to provide aPCR reaction vessel capable of suitably preventing contamination and aPCR device and a PCR method in which the PCR reaction vessel is used.

A PCR reaction vessel according to one embodiment of the presentinvention includes: a substrate; a channel formed on the substrate; apair of filters provided at respective ends of the channel; a pair ofair communication ports that communicate with the channel through therespective filters; a thermal cycle region formed between the pair offilters in the channel; a branch point formed between the pair offilters in the channel; a branched channel whose one end is connected tothe branch point; and a sample introduction port formed at the other endof the branched channel.

Another embodiment of the present invention also relates to a PCRreaction vessel. This PCR reaction vessel includes: a substrate; achannel formed on the substrate; a pair of filters provided atrespective ends of the channel; a pair of air communication ports thatcommunicate with the channel through the respective filters; a thermalcycle region formed between the pair of filters in the channel; a firstbranch point formed between the pair of filters in the channel; a firstbranched channel whose one end is connected to the first branch point; afirst sample introduction port formed at the other end of the firstbranched channel; a second branch point formed between the pair offilters in the channel; a second branched channel whose one end isconnected to the second branch point; and a second sample introductionport formed at the other end of the second branched channel.

The PCR reaction vessel may further include a buffer channel regionformed between the first branch point and the second branch point in thechannel.

The buffer channel region may be set to have a predetermined volumeaccording to the amount of a sample on which a PCR process is intendedto be performed.

The thermal cycle region may include a serpentine channel. The thermalcycle region may be provided with a pair of reaction regions eachincluding a serpentine channel and with a connection region connectingthe pair of reaction regions.

A sealing film for sealing the air communication ports and the sampleintroduction ports may be further provided.

The sealing film may be formed such that the sealing film can beperforated by a needle.

Another embodiment of the present invention relates to a PCR device.This device may includes: the above-described PCR reaction vessel; atemperature adjustment unit for adjusting the temperature of the thermalcycle region; and a pump system that controls the pressure inside thechannel via the air communication ports in order to move the sampleinside the thermal cycle region.

The pump system may be provided with an air pump of a type that allowsthe pressure on a primary side and the pressure on a secondary side tobe equal to each other when stopped.

The air pump may be provided with a nozzle with a hollow needle providedat the tip of the nozzle.

The PCR device may further include a fluorescence detector for detectingfluorescence generated from the sample inside the channel.

The PCR device may further include a control unit for controlling thepump system based on a value detected by the fluorescence detector.

Still another embodiment of the present invention relates to a PCRmethod. This method includes: preparing a PCR reaction vessel including:a substrate; a channel formed on the substrate; a pair of filtersprovided at respective ends of the channel; a pair of air communicationports that communicate with the channel through the respective filters;a thermal cycle region formed between the pair of filters in thechannel; a branch point formed between the pair of filters in thechannel; a branched channel whose one end is connected to the branchpoint; and a sample introduction port formed at the other end of thebranched channel; introducing a sample into the PCR reaction vessel viathe sample introduction port; setting the PCR reaction vessel in a PCRdevice provided with a pump; connecting a nozzle of the pump to the aircommunication ports; and moving a sample in the thermal cycle region bycontrolling the pressure inside the channel by the pump.

A sample not subjected to PCR may stay in the branched channel in themoving of a sample.

Still another embodiment of the present invention also relates to a PCRmethod. This PCR method includes: preparing a PCR reaction vesselincluding: a substrate; a channel formed on the substrate; a pair offilters provided at respective ends of the channel; a pair of aircommunication ports that communicate with the channel through therespective filters; a thermal cycle region formed between the pair offilters in the channel; a first branch point formed between the pair offilters in the channel; a first branched channel whose one end isconnected to the first branch point; a first sample introduction portformed at the other end of the first branched channel; a second branchpoint formed between the pair of filters in the channel; a secondbranched channel whose one end is connected to the second branch point;and a second sample introduction port formed at the other end of thesecond branched channel; introducing a sample into the PCR reactionvessel via the first sample introduction port and the second sampleintroduction port; setting the PCR reaction vessel in a PCR deviceprovided with a pump; connecting a nozzle of the pump to the aircommunication ports; and moving a sample in the thermal cycle region bycontrolling the pressure inside the channel by the pump.

The PCR reaction vessel may further include a buffer channel regionformed between the first branch point and the second branch point in thechannel, and the above-described PCR method may further includes:dispensing the sample using the buffer channel region.

A sample not subjected to PCR may stay in the first branched channel andthe second branched channel in the moving of a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, withreference to the accompanying drawings which are meant to be exemplary,not limiting, and wherein like elements are numbered alike in severalFigures, in which:

FIGS. 1A and 1B are diagrams for explaining a PCR reaction vesselaccording to a first embodiment of the present invention;

FIG. 2 is a cross-sectional view of the PCR reaction vessel shown inFIG. 1A that is sectioned along line A-A;

FIG. 3 is a cross-sectional view of the PCR reaction vessel shown inFIG. 1A that is sectioned along line B-B;

FIG. 4 is a plan view of a substrate provided in the PCR reaction vesselaccording to the first embodiment;

FIG. 5 is a conceptual diagram for explaining the configuration of thePCR reaction vessel according to the first embodiment;

FIG. 6 is a diagram schematically showing a condition where a sample isintroduced into the PCR reaction vessel in the first embodiment;

FIG. 7 is a diagram showing a state where a third sealing film isreattached to the substrate in the first embodiment;

FIG. 8 is a diagram for explaining a PCR device in which the PCRreaction vessel according to the first embodiment is used;

FIG. 9 is a diagram for explaining a state where the PCR reaction vesselis set at a predetermined position of the PCR device in the firstembodiment;

FIG. 10 is a diagram showing a condition where a nozzle of a pump systemand an air communication port of the PCR reaction vessel are connectedin the first embodiment;

FIG. 11 is a cross-sectional view of the PCR reaction vessel shown inFIG. 10 that is sectioned along line C-C;

FIG. 12 is a diagram showing a condition where the pump system isoperated so as to move the sample in the first embodiment;

FIGS. 13A and 13B are diagrams for explaining a PCR reaction vesselaccording to a second embodiment of the present invention;

FIG. 14 is a cross-sectional view of the PCR reaction vessel shown inFIG. 13A that is sectioned along line A-A;

FIG. 15 is a cross-sectional view of the PCR reaction vessel shown inFIG. 13A that is sectioned along line B-B;

FIG. 16 is a plan view of a substrate provided in the PCR reactionvessel according to the second embodiment;

FIG. 17 is a conceptual diagram for explaining the configuration of thePCR reaction vessel according to the second embodiment;

FIG. 18 is a diagram schematically showing a condition where a sample isintroduced into the PCR reaction vessel in the second embodiment;

FIG. 19 is a diagram showing a state where a third sealing film isreattached to the substrate in the second embodiment;

FIG. 20 is a diagram for explaining a PCR device in which the PCRreaction vessel according to the second embodiment is used;

FIG. 21 is a diagram for explaining a state where the PCR reactionvessel is set at a predetermined position of the PCR device in thesecond embodiment;

FIG. 22 is a diagram showing a condition where a nozzle of a pump systemand an air communication port of the PCR reaction vessel are connectedin the second embodiment;

FIG. 23 is a cross-sectional view of the PCR reaction vessel shown inFIG. 22 that is sectioned along line C-C; and

FIG. 24 is a diagram showing a condition where the pump system isoperated so as to move the sample in the second embodiment.

DETAILED DESCRIPTION OF THE INVENTION

An explanation will be given in the following regarding a PCR reactionvessel and a PCR device according to an embodiment of the presentinvention. The same or equivalent constituting elements, members, andprocesses illustrated in each drawing shall be denoted by the samereference numerals, and duplicative explanations will be omittedappropriately. Reference herein to details of the illustratedembodiments is not intended to limit the scope of the claims. It shouldbe understood that not all of the features and the combination thereofdiscussed are essential to the invention.

First Embodiment

FIGS. 1A and 1B are diagrams for explaining a PCR reaction vessel 10according to a first embodiment of the present invention. FIG. 1A is aplan view of the PCR reaction vessel 10, and FIG. 1B is a front view ofthe PCR reaction vessel 10. FIG. 2 is a cross-sectional view of the PCRreaction vessel 10 shown in FIG. 1A that is sectioned along line A-A.FIG. 3 is a cross-sectional view of the PCR reaction vessel 10 shown inFIG. 1A that is sectioned along line B-B. FIG. 4 is a plan view of asubstrate 14 provided in the PCR reaction vessel 10. FIG. 5 is aconceptual diagram for explaining the configuration of the PCR reactionvessel 10.

The PCR reaction vessel 10 comprises a resinous substrate 14 having agroove-like channel 12 formed on a lower surface 14 a thereof, a channelsealing film 16, which is attached on the lower surface 14 a of thesubstrate 14, for sealing the channel 12, and three sealing films (afirst sealing film 18, a second sealing film 20, and a third sealingfilm 22) attached on an upper surface 14 b of the substrate 14.

The substrate 14 is preferably formed of a material that has goodthermal conductivity, is stable under temperature changes, and isresistant to a sample solution that is used. Further, the substrate 14is preferably formed of a material that has good moldability, a goodtransparency and barrier property, and a low self-fluorescence property.As such a material, an inorganic material such as glass, silicon, or thelike, a resin such as acrylic, polyester, silicone, or the like, andparticularly cycloolefin are preferred. An example of the dimensions ofthe substrate 14 includes a long side of 70 mm, a short side of 42 mm,and a thickness of 3 mm. An example of the dimensions of the channel 12formed on the lower surface 14 a of the substrate 14 includes a width of0.5 mm and a depth of 0.5 mm.

As described above, the groove-like channel 12 is formed on the lowersurface 14 a of the substrate 14, and this channel 12 is sealed by thechannel sealing film 16 (see FIG. 2 ). A first air communication port 24is formed at the position of one end 12 a of the channel 12 in thesubstrate 14. A second air communication port 26 is formed at theposition of the other end 12 b of the channel 12 in the substrate 14.The pair, the first air communication port 24 and the second aircommunication port 26, is formed so as to be exposed on the uppersurface 14 b of the substrate 14. Such a substrate can be produced byinjection molding or cutting work with an NC processing machine or thelike.

A first filter 28 is provided between the first air communication port24 and one end 12 a of the channel 12 in the substrate 14 (see FIG. 2 ).A second filter 30 is provided between the second air communication port26 and the other end 12 b of the channel 12 in the substrate 14. Thepair, the first filter 28 and the second filter 30, provided atrespective ends of the channel 12, has good low impurity characteristicsand also allows only air to pass therethrough so as to preventcontamination such that the quality of DNA amplified by PCR does notdeteriorate. As a filter material, polyethylene, PTFE, and the like aresuitable, and the filter material may be porous or hydrophobic.Regarding the dimensions of the first filter 28 and the second filter30, the first filter 28 and the second filter 30 are formed so as to fitwithout any gap in a filter installation space formed in the substrate14.

At a branch point 112 c located between the first filter 28 and thesecond filter 30, a branched channel 131 branching from the channel 12is formed in the substrate 14. A sample introduction port 133 is formedat the position of a terminal end 31 a of the branched channel 131 inthe substrate 14 (see FIG. 3 ). The sample introduction port 133 isformed so as to be exposed on the upper surface 14 b of the substrate14.

A section of the channel 12 that is located between the first filter 28and the branch point 112 c forms a thermal cycle region 12 e intendedfor a high temperature region and a medium temperature region in orderto apply a thermal cycle to the sample. The thermal cycle region 12 e ofthe channel 12 includes a serpentine channel. This is for efficientlyproviding the amount of heat provided from the PCR device during a PCRstep to a sample and for allowing the volume of a sample that can besubjected to PCR to be a certain amount or more. In the firstembodiment, the branch point 112 c is provided between the thermal cycleregion 12 e and the second filter 30. However, since the branch point112 c is for introducing a sample subjected to PCR through the branchedchannel 131 and the sample introduction port 133, which are connected tothe branch point 112 c, there is no functional problem as long as thebranch point 112 c is formed between the first filter 28 and the secondfilter 30. Since the PCR reaction vessel 10 is intended to be installedin the PCR device, to provide a thermal cycle to a sample, and tomeasure optical properties such as fluorescence emitted from the sample,the arrangement of each element including the channels and branch pointneeds to be arbitrarily selected also in consideration of, e.g., thearrangement of a temperature adjustment unit and a probe forfluorescence detection, which are described later. In the firstembodiment, the branch point 112 c is arranged on the side closer to thesecond filter 30, and the thermal cycle region is provided between thebranch point 112 c and the first filter 28. Therefore, the distance onthe channel between the branch point 112 c and the first filter 28 canbe kept to be relatively large, and a space for efficiently arrangingthe temperature adjustment unit is formed, when installed in the thermalcycle region and also in the PCR device. On the contrary, when thebranch point 112 c is arranged on the side closer to the first filter28, it can be considered to be more reasonable to form the thermal cycleregion 12 e between the branch point 112 c and the second filter 30.

In the PCR reaction vessel 10 according to the first embodiment, most ofthe channel 12 is formed in the shape of a groove exposed on the lowersurface 14 a of the substrate 14. This is for allowing for easiermolding by injection molding using a metal mold or the like. In order tomake use of this groove as a channel, the channel sealing film 16 isattached on the lower surface 14 a of the substrate 14. The channelsealing film 16 may be sticky on one of the main surfaces thereof or mayhave a functional layer that exhibits stickiness or adhesiveness bypressing that is formed on one of the main surfaces. Thus, the channelsealing film 16 has a function of being easily able to become integralwith the lower surface 14 a of the substrate 14 while being in closecontact with the lower surface 14 a. The channel sealing film 16 isdesirably formed of a material, including an adhesive, that has a lowself-fluorescence property. In this respect, a transparent film made ofa resin such as a cycloolefin polymer, polyester, polypropylene,polyethylene or acrylic is suitable but is not limited thereto. Further,the channel sealing film 16 may be formed of a plate-like glass orresin. Since rigidity can be expected in this case, the channel sealingfilm 16 is useful for preventing warpage and deformation of the PCRreaction vessel 10.

Further, in the PCR reaction vessel 10 according to the firstembodiment, the first air communication port 24, the second aircommunication port 26, the first filter 28, the second filter 30, andthe sample introduction port 133 are exposed on the upper surface 14 bof the substrate 14. Therefore, in order to seal the first aircommunication port 24 and the first filter 28, the first sealing film 18is attached to the upper surface 14 b of the substrate 14. Also, inorder to seal the second air communication port 26 and the second filter30, the second sealing film 20 is attached to the upper surface 14 b ofthe substrate 14. Further, in order to seal the sample introduction port133, the third sealing film 22 is attached to the upper surface 14 b ofthe substrate 14.

The first sealing film 18 that is used has a size that is capable ofsealing the first air communication port 24 and the first filter 28 atthe same time, and the second sealing film 20 that is used has a sizethat is capable of sealing the second air communication port 26 and thesecond filter 30 at the same time. A pressure-type pump (describedlater) is connected to the first air communication port 24 and thesecond air communication port 26 by perforating the first aircommunication port 24 and the second air communication port 26 by ahollow needle (syringe needle with a sharp tip) provided at the tip ofthe pump. Therefore, the first sealing film 18 and the second sealingfilm 20 are preferably films made of a material that is easilyperforated by the needle and/or have a thickness that is easilyperforated by the needle. In the first embodiment, sealing films aredescribed that each has a size that allows for the sealing of an aircommunication port and a filter that are concerned at the same time.However, these air communication port and filter may be sealedseparately. Alternatively, a sealing film may be used that can seal thefirst air communication port 24, the first filter 28, the second aircommunication port 26, and the second filter 30 all at once (by a singlesheet).

As the third sealing film 22, a sealing film having a size that allowsfor the sealing of the sample introduction port 133 is used.Introduction of a sample into the channel 12 through the sampleintroduction port 133 is performed by once peeling the third sealingfilm 22 from the substrate 14, and, after the introduction of apredetermined amount of sample, the third sealing film 22 is put backbeing attached to the upper surface 14 b of the substrate 14 again.Therefore, as the third sealing film 22, a film is desired that issticky enough to hold up through several cycles of attaching andpeeling. Alternatively, as the third sealing film 22, a new film may beattached after the introduction of a sample. In this case, theimportance of the property related to attaching and peeling can belessened.

Also, at the time of the introduction of a sample, it is necessary toonce peel off either the first sealing film 18 or the second sealingfilm 20 in the same way as in the third sealing film 22. This is becausethe sample does not enter the channel if an air outlet is not created.Therefore, the first sealing film 18 and the second sealing film 20 aredesirably films that are sticky enough to hold up through several cyclesof attaching and peeling. Alternatively, a new film may be attachedafter the introduction of a sample.

In the same way as in the channel sealing film 16, the first sealingfilm 18, the second sealing film 20, and the third sealing film 22 mayhave an adhesive layer or a functional layer exhibiting stickiness oradhesiveness by pressing that is formed on one of the main surfacesthereof. The first sealing film 18, the second sealing film 20, and thethird sealing film 22 are desirably formed of a material, including anadhesive, that has a low self-fluorescence property. In this respect, atransparent film made of a resin such as cycloolefin, polyester,polypropylene, polyethylene or acrylic is suitable but is not limitedthereto. As described above, the property such as stickiness or the likedesirably do not degrade to such an extent that the use is affected evenafter attaching and peeling of multiple times. However, in a case wherea new film is attached after the peeling and the introduction of asample or the like, the importance of this property related to theattaching and peeling can be lessened.

An explanation will be given next regarding a method of using the PCRreaction vessel 10 configured as described above. First, a sample to beamplified through a thermal cycle is prepared. The sample includes thoseobtained by adding a plurality of types of primers, a thermostableenzyme and four types of deoxyribonucleoside triphosphates (dATP, dCTP,dGTP, dTTP) as PCR reagents to a mixture containing two or more types ofDNA. Next, the first sealing film 18 and the third sealing film 22 arepeeled off from the substrate 14 such that the first air communicationport 24 and the sample introduction port 133 are open. In the case wherethe first sealing film 18 is of a size that is capable of sealing thefirst air communication port 24 and the first filter 28 at the sametime, the first sealing film 18 may be completely peeled from thesubstrate 14 such that the first air communication port 24 and the firstfilter 28 are open to the atmospheric air. However, opening only thefirst air communication port 24 without completely peeling the firstsealing film 18 from the substrate 14 is effective in the prevention ofcontamination since the first filter 28 is not exposed to theatmospheric air. Also, in the case of using a sealing film capable ofseparately sealing the first air communication port 24 and the firstfilter 28, the first filter 28 is not exposed to the atmospheric air inthe same manner, and the film is thus effective in the prevention ofcontamination.

The sample is then introduced to the sample introduction port 133 by adropper, a syringe, or the like. FIG. 6 schematically shows a conditionwhere a sample 70 is introduced into the PCR reaction vessel 10. In FIG.6 , in order to emphasize the position of the sample 70, the sample 70is shown by a solid line that is thicker than that for the channel 12.It should be noted that the solid line does not indicate a state wherethe sample 70 overflows outside the channel.

As shown in FIG. 6 , the sample 70 introduced to the sample introductionport 133 fills the channel by being pushed in by a dropper, a syringe,or the like, or by a capillary phenomenon. The sample 70 is packed inthe direction of the thermal cycle region 12 e (in the direction of thefirst air communication port 24) beyond the branch point 112 c in thechannel 12. However, the sample 70 is not packed in the direction of thesecond air communication port 26 beyond the branch point 112 c. This isbecause the second air communication port 26 is sealed such that thereis no outlet for the air to escape.

Next, as shown in FIG. 7 , the first sealing film 18 and the thirdsealing film 22 are attached to the substrate 14 again so as to seal thefirst air communication port 24 and the sample introduction port 133. Asdescribed above, a new first sealing film 18 and a new third sealingfilm 22 may be attached. This completes the introduction of the sample70 into the PCR reaction vessel 10.

FIG. 8 is a diagram for explaining a PCR device 100 in which the PCRreaction vessel 10 is used. FIG. 9 is a diagram for explaining a statewhere the PCR reaction vessel 10 is set at a predetermined position ofthe PCR device 100.

The PCR device 100 is provided with a fluorescence detection opticalprobe 122, a first heater 134, and a second heater 135. As shown in FIG.9 , in the PCR reaction vessel 10, two reaction regions of the thermalcycle region 12 e of the channel 12 are arranged on the first heater 134and the second heater 135, respectively, and the fluorescence detectionoptical probe 122 is installed in the PCR device 100 so as to be locatedin a connection region between the two reaction regions.

The PCR device 100 is further provided with a pump system 110 forcausing the sample 70 to reciprocate in the thermal cycle region 12 e.This pump system 110 is provided with a first nozzle 101, a secondnozzle 102, a first pump 103, a second pump 104, a first pump driver105, a second pump driver 106, and a control unit 107. The first nozzle101 of the pump system 110 is connected to the first air communicationport 24 of the PCR reaction vessel 10, and the second nozzle 102 of thepump system 110 is connected to the second air communication port 26 ofthe PCR reaction vessel 10. A specific method for connecting the nozzlesto the respective air communication ports will be described later. Thepump system 110 moves the sample in the thermal cycle region 12 e bycontrolling the pressure inside the channel 12 via the first aircommunication port 24 and the second air communication port 26.

In the PCR device 100 according to the first embodiment, the firstheater 134 and the second heater 135 are set to different temperatures.Each heater provides the amount of heat to individually control thetemperatures of the two reaction regions in the thermal cycle region 12e and has an area that covers the area of each of the reaction regions.Also, each heater may be a means or a structure such as resistiveheating or a Peltier element. For example, the first heater 134 iscontrolled by the first heater driver 130 so as to maintain thetemperature of the reaction region on the right side of the figure pagein the thermal cycle region 12 e of the channel 12 to be 94° C.constantly. Also, the second heater 135 is controlled by the secondheater driver 132 so as to maintain the temperature of the reactionregion on the left side of the figure page to be 60° C. constantly. Thetemperature of each reaction region may be measured by a temperaturesensor (not shown) such as a thermocouple, and the output to each heatermay be controlled by each driver based on an electric signal therefrom.In this manner, the first heater 134, the second heater 135, the firstheater driver 130, the second heater driver 132, and the temperaturesensor may constitute a temperature adjustment unit for adjusting thetemperature of the thermal cycle region 12 e and may include otherelements that improve the controllability of the temperature. In thefollowing, the reaction region at the atmospheric temperature of 94° C.in the channel 12 is referred to as “high temperature part 111”, and thereaction region at the atmospheric temperature of 60° C. in the channel12 is referred to as “medium temperature part 112”. Further, in thepresent embodiment, a detailed explanation will be given regarding a PCRdevice that is provided with a PCR reaction vessel provided with athermal cycle region where temperature ranges of two levels are set astwo reaction regions and that is provided with a temperature controlunit. However, the PCR device may be provided with a PCR reaction vesselprovided with a thermal cycle region where temperature ranges of threeor more levels can be set and that is provided with a temperaturecontrol unit. In this case, (although not shown), for example, the PCRdevice may be provided with a PCR reaction vessel provided with reactionregions in which a low temperature part, a medium temperature part, anda high temperature part are arranged from the left side of the figurepage and with a temperature control unit. In such a case, for example,the low temperature part, the medium temperature part, and the hightemperature part are controlled to maintain 50 to 70° C., at 72° C., and94° C., respectively.

The pump system 110 is arranged to cause the sample 70 to reciprocatewithin the thermal cycle region 12 e of the channel 12, as describedabove. By alternately operating the first pump 103 and the second pump104 through the first pump driver 105 and the second pump driver 106under a certain condition by the control unit 107, the sample 70 can bereciprocated between the high temperature part 111 and the mediumtemperature part 112 of the channel 12, and a thermal cycle can beapplied to the sample 70 under a certain condition. In the PCR device100 according to the first embodiment, the first pump 103 and the secondpump 104 are air pumps or blower pumps of a type where, when both thefirst pump 103 and the second pump 104 are stopped, the atmosphericpressures on a primary side and a secondary side instantaneously becomeequal to each other, and when both first pump 103 and the second pump104 are being stopped, the atmospheric pressure on the primary side andthe atmospheric pressure on the secondary side are equal to each other.If this type of pump is not used, that is, if a pump is used thatmaintains the immediately preceding pressure even when stopped, there isa possibility that a phenomenon occurs where the sample continues tomove slightly even when the pump is stopped such that the sample doesnot stop in a predetermined reaction region and the temperature of thesample cannot be appropriately controlled. On the other hand, in the PCRdevice 100 according to the first embodiment, external air and thechannel of the PCR reaction vessel communicate with each other in termsof the atmospheric pressure when stopped (when opened), having equalatmospheric pressure; however, since a filter is provided between theair communication port and the channel, contamination into the channelcan be prevented.

The sample 70 can undergo PCR by the above-described thermal cycle, andthe fluorescence from the sample 70 in the channel can be detected, andthe value thereof can be used as an index serving as information fordetermining the progress of the PCR or the termination of the reaction.As the fluorescence detection optical probe 122 and the driver 121,optical fiber-type fluorescence detector FLE-510 (manufactured by NipponSheet Glass Co., Ltd.) can be used, which is a very compact opticalsystem that allows for rapid measurement and the detection offluorescence regardless of a light and/or dark atmosphere. This opticalfiber type fluorescence detector can be also arranged easily in a narrowspace between the two reaction regions in the thermal cycle region. Thisoptical fiber type fluorescence detector allows the wavelengthcharacteristic of the excitation light/fluorescence to be tuned suchthat the wavelength characteristic is suitable for the fluorescencecharacteristic of the sample 70 and thus allows an optimum optical anddetection system for a sample having various characteristics to beprovided. Further, fluorescence detection optical probes 122 and drivers121 may be provided that are installed at a plurality of sitesthroughout the inside of the thermal cycle region 12 e. For example, thefluorescence detection optical probes 122 and drivers 121 may beinstalled to detect the fluorescence from the sample 70 in the channellocated in the high temperature part 111 or the medium temperature part112. In addition to the function of acquiring information fordetermining the progress or the termination of the PCR, the fluorescencedetection optical probes 122 and drivers 121 can also cause to afunction as position sensors for detecting, without fail, whether or notthe sample 70 is in the high temperature part 111 or the mediumtemperature part 112.

In the PCR device 100 configured as described above, the control unit107 of the pump system 110, the driver 121 of the fluorescence detectionoptical probe 122, the first heater driver 130, and the second heaterdriver 132 are controlled to operate optimally by a CPU 141. Also, asdescribed above, in the case where a reaction region in whichtemperatures of three levels are set, a third heater driver (not shown)is also controlled by the CPU in addition to the above.

FIG. 10 is a diagram showing a condition where a nozzle of a pump systemand an air communication port of the PCR reaction vessel are connected.FIG. 11 is a cross-sectional view of the PCR reaction vessel 10 shown inFIG. 10 that is sectioned along line C-C. As described above, the firstnozzle 101 is connected to the first air communication port 24, and thesecond nozzle 102 is connected to the second air communication port 26.

As shown in FIG. 11 , a hollow needle 150 is provided at the tip of thefirst nozzle 101. By perforating the first sealing film 18 with thisneedle 150, the first nozzle 101 is connected to the first aircommunication port 24. The same applies to the connection between thesecond nozzle 102 and the second air communication port 26.

The needle 150 is provided with a packing material 151 made of a softresin that comes into close contact with the surface of a sealing filmin order to secure airtightness around the connection. Immediately afterthe PCR reaction vessel 10 is set in the PCR device 100, the pump system110 is not in operation and is open to the atmospheric air, and thepressure inside the channel is thus in a state where the pressure isequal to the atmospheric pressure.

FIG. 12 shows a condition where the pump system 110 is operated so as tomove the sample 70. Either one of the first pump 103 and the second pump104 is operated so as to move the sample 70 to the high temperature part111 or the medium temperature part 112 of the thermal cycle region 12 e.In FIG. 12 , the second pump 104 to which the second nozzle 102 isconnected is operated, and the first pump 103 to which the first nozzle101 is connected is stopped. In other words, the first air communicationport 24 to which the first nozzle 101 extending from the first pump 103is connected is open to the atmospheric pressure. When the second pump104 is operated to feed the air from the second nozzle 102 to the secondair communication port 26, the sample 70 moves, passing through themedium temperature part 112 and moving to the high temperature part 111.This state is assumed to be an initial state.

More specifically, at the start of the operation of the second pump 104or immediately before the start of the operation of the second pump 104,monitoring of the fluorescence emitted from the sample in the channel isstarted using the fluorescence detection optical probe 122. When thereis nothing at a measurement point of the fluorescence detection opticalprobe 122, fluorescence that is detected is at zero or at a backgroundlevel. When the sample 70 exists at the measurement point, fluorescenceis detected. Therefore, monitoring of the fluorescence is started beforethe operation of the second pump 104 starts, the completion of themovement of the sample 70 to the high temperature part 111 is recognizedthrough a fluorescence value rising from the background level anddropping to the background level again, and stopping the operation ofthe second pump 104 at this point completes the setting of the initialstate. Also, when the fluorescence detection optical probe is furtherlocated in the high temperature part 111, it is also possible to stopthe sample 70 at the high temperature part 111 more certainly.

It should be noted that the sample 70 located inside the branchedchannel 131 stays around the place even when the second pump 104 isoperated. This is because the sample introduction port 133 is sealedwith the third sealing film 22. The sample 70 located inside thebranched channel 131 is not subjected to PCR.

After the setting to the initial state, a thermal cycle is applied tothe sample 70 to progress the PCR. The measurement of fluorescence bythe fluorescence detection optical probe 122 is continued.

(A) First, the sample 70 is allowed to sit for 1 to 30 seconds in thehigh temperature part 111 (about 94° C. atmosphere) (denaturation:thermal denaturation step). Through this step, double-stranded DNA isdenatured into single strands.

(B) Next, the first pump 103 to which the first nozzle 101 is connectedis operated to move the sample 70 to the medium temperature part 112(about 60° C. atmosphere). More specifically, the sample 70 is pushed ina direction from the high temperature part 111 to the medium temperaturepart 112 by the action of the first pump 103. Since the fluorescencemeasurement by the fluorescence detection optical probe 122 continues,the operation of the first pump 103 is stopped at the point of time whena fluorescence amount rises from the background level and drops againdue to the sample 70 passing through the measurement point of thefluorescence detection optical probe 122 (or after a certain period oftime has passed after the fluorescence amount has decreased). Further,when the fluorescence detection optical probe 122 is located at themedium temperature part 112, it is also possible to more certainly stopthe sample 70 at the medium temperature part 112.

(C) In the medium temperature part 112, the sample 70 is allowed to sitfor 3 to 60 seconds (annealing+elongation: annealing step+elongationstep). Through these steps, binding of primers contained in the sample70 in advance occurs resulting in further elongated DNA.

(D) Next, the second pump 104 to which the second nozzle 102 isconnected is operated to move the sample 70 from the medium temperaturepart 112 to the high temperature part 111. The timing for stopping thepump operation is determined based on changes in the fluorescence amountmeasured by the fluorescence detection optical probe 122 in the samemanner as described above. After moving the sample 70 to the hightemperature part 111, the sample 70 is allowed to sit for 1 to 30seconds to go through heat denaturation.

(E) By repeating the above (B) to (D) for a predetermined number ofcycles, applying a thermal cycle to the sample 70, and allowing the DNAcontained in the sample 70 to undergo a plurality of cycles of thermaldenaturation, annealing, and elongation steps, the amplification of DNAis performed. The number of cycles is appropriately determined by acombination of target DNA, primers, enzymes, and the like.

After the completion of a predetermined number of thermal cycles, thefirst pump 103 and the second pump 104 are stopped, and the PCR isended. Even when the predetermined number of thermal cycles are applied,the fluorescence is measured by the fluorescence detection optical probe122, and the fluorescence detected from the sample 70 increases as theDNA contained in the sample 70 is amplified. Thereby, the concentrationof the sample 70 can be accurately known.

According to the PCR reaction vessel 10 according to the firstembodiment, by providing the first filter 28 between the first aircommunication port 24 and the channel 12 and the second filter 30between the second air communication port 26 and the channel 12,contamination inside the channel 12 can be prevented. Although theimplementation of measures to prevent contamination on the side of thepump system 110 is likely to increase the cost, in the PCR reactionvessel 10 according to the first embodiment, the measures allowcontamination to be prevented only on the PCR reaction vessel 10 sideand are thus economical. Further, when the PCR reaction vessel is usedas a disposable vessel, since the filter is always a new one,contamination can be further prevented at low cost. Furthermore,regarding the disposal of the PCR reaction vessel, since the sample issubstantially sealed in the PCR reaction vessel, the disposal is alsomeaningful in terms of safety and environment.

In the PCR device 100 according to the first embodiment, the sample canbe caused to reciprocate inside the channel 12 of the PCR reactionvessel 10 by alternately operating the first pump 103 and the secondpump 104 that allow the pressures on the primary side and the secondaryside to become equal when stopped. In this case, since excessivepressure is not applied to the sample during the liquid feeding(applying pressure to the sample in the channel) and, further, thepressure in the channel is not reduced, evaporation and boiling(foaming) of the liquid containing the sample due to the action of thehigh temperature part 111 can be prevented.

Further, in the PCR device 100 according to the first embodiment, thefluorescence from the sample is monitored all the time even during PCRin the thermal cycle region (real-time PCR). As a result, the end timingof the PCR can be determined based on the fluorescence amount that hasbeen measured. Further, by monitoring a change in fluorescence by thefluorescence detection optical probe 122, the passing of the sample canbe known, and, based on the change in the fluorescence amountaccompanying the passing of the sample, the alternate operation of thefirst pump 103 and the second pump 104 can be controlled. Thus, thesample to be subjected to the PCR can be accurately positioned to thehigh temperature part 111 or the medium temperature part 112 of thethermal cycle region.

On the other hand, in the case of a PCR reaction vessel and a PCR devicehaving a reaction region where the above-described temperatures of threelevels: the high temperature part; medium temperature part; and lowtemperature part, are controlled, the steps, heat denaturation,annealing, and elongation, can be performed at the high temperaturepart, at the medium temperature part, and at the low temperature part,respectively. Also, the control thereof can be easily developed andimproved by those skilled in the art based on the above detaileddescription. Those skilled in the art can appropriately choose,depending on the characteristics of the sample, whether the reactionregion is set to have two levels or three levels.

Second Embodiment

FIGS. 13A and 13B are diagrams for explaining a PCR reaction vessel 210according to a second embodiment of the present invention. FIG. 13A is aplan view of the PCR reaction vessel 210, and FIG. 13B is a front viewof the PCR reaction vessel 210. FIG. 14 is a cross-sectional view of thePCR reaction vessel 210 shown in FIG. 13A that is sectioned along lineA-A. FIG. 15 is a cross-sectional view of the PCR reaction vessel 210shown in FIG. 13A that is sectioned along line B-B. FIG. 16 is a planview of a substrate 214 provided in the PCR reaction vessel 210. FIG. 17is a conceptual diagram for explaining the configuration of the PCRreaction vessel 210. The PCR reaction vessel 210 in the secondembodiment is different from that in the first embodiment in that thePCR reaction vessel is provided with two branch points (a first branchpoint 212 c and a second branch point 212 d), two branched channels andtwo sample introduction ports extended from the branch points (a firstbranched channel 231 and a first sample introduction port 233, a secondbranched channel 232 and a second sample introduction port 234), and abuffer channel region 212 f between the first branch point 212 c and thesecond branch point 212 d.

The PCR reaction vessel 210 comprises a resinous substrate 214 having agroove-like channel 212 formed on a lower surface 214 a thereof, achannel sealing film 216, which is attached on the lower surface 214 aof the substrate 214, for sealing the channel 212, and three sealingfilms (a first sealing film 218, a second sealing film 220, and a thirdsealing film 222) attached on an upper surface 214 b of the substrate214.

The substrate 214 is preferably formed of a material that has goodthermal conductivity, is stable against temperature change, and isresistant to a sample solution that is used. Further, the substrate 214is preferably formed of a material that has good moldability, goodtransparency and barrier property, and low self-fluorescence property.As such a material, an inorganic material such as glass, silicon, or thelike, a resin such as acrylic, polyester, silicone, or the like, and,particularly, cycloolefin are preferred. An example of the dimensions ofthe substrate 214 includes a long side of 70 mm, a short side of 42 mm,and a thickness of 3 mm. An example of the dimensions of the channel 212formed on the lower surface 214 a of the substrate 214 includes a widthof 0.5 mm and a depth of 0.5 mm.

As described above, the groove-like channel 212 is formed on the lowersurface 214 a of the substrate 214, and this channel 212 is sealed bythe channel sealing film 216 (see FIG. 14 ). A first air communicationport 224 is formed at the position of one end 212 a of the channel 212in the substrate 214. A second air communication port 226 is formed atthe position of the other end 212 b of the channel 212 in the substrate214. The pair, the first air communication port 224 and the second aircommunication port 226, is formed so as to be exposed on the uppersurface 214 b of the substrate 214. Such a substrate can be produced byinjection molding or cutting work with an NC processing machine or thelike.

A first filter 228 is provided between the first air communication port224 and one end 212 a of the channel 212 in the substrate 214 (see FIG.14 ). A second filter 230 is provided between the second aircommunication port 226 and the other end 212 b of the channel 212 in thesubstrate 214. The pair, the first filter 228 and the second filter 230,provided at respective ends of the channel 212 has good low impuritycharacteristics and also allows only air to pass therethrough so as toprevent contamination such that the quality of DNA amplified by PCR doesnot deteriorate. As a filter material, polyethylene, PTFE, and the likeare suitable, and the filter material may be porous or hydrophobic.Regarding the dimensions of the first filter 228 and the second filter230, the first filter 228 and the second filter 230 are formed so as tofit without any gap in a filter installation space formed in thesubstrate 214.

At a first branch point 212 c located between the first filter 228 andthe second filter 230, a first branched channel 231 branching from thechannel 212 is formed in the substrate 214. A first sample introductionport 233 is formed at the position of a terminal end 231 a of the firstbranched channel 231 in the substrate 214 (see FIG. 15 ). At a secondbranch point 212 d located between the first branch point 212 c and thesecond filter 230, a second branched channel 232 branching from thechannel 212 is further formed in the substrate 214. A second sampleintroduction port 234 is provided at the position of a terminal end 232a of the second branched channel 232 in the substrate 214. The firstsample introduction port 233 and the second sample introduction port 234are formed so as to be exposed on the upper surface 214 b of thesubstrate 214.

A section of the channel 212 that is located between the first filter228 and the first branch point 212 c forms a thermal cycle region 212 eintended for a high temperature region and a medium temperature regionin order to apply a thermal cycle to the sample. The thermal cycleregion 212 e of the channel 212 includes a serpentine channel. This isfor efficiently providing the amount of heat provided from the PCRdevice during a PCR step to a sample and for allowing the volume of asample that can be subjected to PCR to be a certain amount or more. Thethermal cycle region 212 e is provided with a pair of reaction regionseach including a serpentine channel and with a connection regionconnecting the pair of reaction regions.

A section of the channel 212 that is located between the first branchpoint 212 c and the second branch point 212 d forms the buffer channelregion 212 f. The buffer channel region 212 f of the channel 212includes a serpentine channel. The volume of the buffer channel region212 f of the channel 212 is set to a predetermined volume according tothe amount of a sample on which a PCR process is intended to beperformed. The function of the buffer channel region will be describedlater.

In the PCR reaction vessel 210 according to the second embodiment, mostof the channel 212 is formed in the shape of a groove exposed on thelower surface 214 a of the substrate 214. This is for allowing foreasier molding by injection molding using a metal mold or the like. Inorder to make use of this groove as a channel, the channel sealing film216 is attached on the lower surface 214 a of the substrate 214. Thechannel sealing film 216 may be sticky on one of the main surfacesthereof or may have a functional layer that exhibits stickiness oradhesiveness by pressing that is formed on one of the main surfaces.Thus, the channel sealing film 216 has a function of being easily ableto become integral with the lower surface 214 a of the substrate 214while being in close contact with the lower surface 214 a. The channelsealing film 216 is desirably formed of a material, including anadhesive, that has a low self-fluorescence property. In this respect, atransparent film made of a resin such as a cycloolefin polymer,polyester, polypropylene, polyethylene or acrylic is suitable but is notlimited thereto. Further, the channel sealing film 216 may be formed ofa plate-like glass or resin. Since rigidity can be expected in thiscase, the channel sealing film 16 is useful for preventing warpage anddeformation of the PCR reaction vessel 210.

Further, in the PCR reaction vessel 210 according to the secondembodiment, the first air communication port 224, the second aircommunication port 226, the first sample introduction port 233, thesecond sample introduction port 234, the first filter 228, and thesecond filter 230 are exposed on the upper surface 214 b of thesubstrate 214. Therefore, in order to seal the first air communicationport 224 and the first filter 228, the first sealing film 218 isattached to the upper surface 214 b of the substrate 214. Therefore, inorder to seal the second air communication port 226 and the secondfilter 230, the second sealing film 220 is attached to the upper surface214 b of the substrate 214. Therefore, in order to seal the first sampleintroduction port 233 and the second sample introduction port 234, thethird sealing film 222 is attached to the upper surface 214 b of thesubstrate 214.

The first sealing film 218 that is used has a size that is capable ofsealing the first air communication port 224 and the first filter 228 atthe same time, and the second sealing film 220 that is used has a sizethat is capable of sealing the second air communication port 226 and thesecond filter 230 at the same time. A pressure-type pump (describedlater) is connected to the first air communication port 224 and thesecond air communication port 226 by perforating the first aircommunication port 224 and the second air communication port 226 by ahollow needle (syringe needle with a sharp tip) provided at the tip ofthe pump. Therefore, the first sealing film 128 and the second sealingfilm 220 are preferably films made of a material that is easilyperforated by the needle and/or have a thickness that is easilyperforated by the needle. In the second embodiment, the sealing filmseach having a size that is capable of sealing corresponding aircommunication port and filter at the same time are described. However,these air communication port and filter may be sealed separately.Alternatively, a sealing film may be used that can seal the first aircommunication port 224, the first filter 228, the second aircommunication port 226, and the second filter 230 all at once (by asingle sheet).

As the third sealing film 222, a sealing film having a size that iscapable of sealing the first sample introduction port 233 and the secondsample introduction port 234 at the same time is used. Introduction of asample into the channel 212 through the first sample introduction port233 and the second sample introduction port 234 is performed by oncepeeling the third sealing film 222 from the substrate 214, and, afterthe introduction of a predetermined amount of sample, the third sealingfilm 222 is put back being attached to the upper surface 214 b of thesubstrate 214 again. Therefore, as the third sealing film 222, a film isdesired that is sticky enough to hold up through several cycles ofattaching and peeling. Alternatively, as the third sealing film 222, anew film may be attached after the introduction of a sample. In thiscase, the importance of the property related to attaching and peelingcan be lessened. In the second embodiment, the sealing films each havinga size that is capable of sealing the first sample introduction port 233and the second sample introduction port 234 at the same time aredescribed. However, these air communication port and filter may besealed separately.

In the same way as in the channel sealing film 216, the first sealingfilm 218, the second sealing film 220, and the third sealing film 222may have an adhesive layer or a functional layer exhibiting stickinessor adhesiveness by pressing that is formed on one of the main surfacesthereof. The first sealing film 218, the second sealing film 220, andthe third sealing film 222 are desirably formed of a material, includingan adhesive, that has a low self-fluorescence property. In this respect,a transparent film made of a resin such as cycloolefin (COP), polyester,polypropylene, polyethylene or acrylic is suitable but is not limitedthereto. As described above, the property such as stickiness or the likedesirably do not degrade to such an extent that the use is affected evenafter attaching and peeling of multiple times. However, in a case wherea new film is attached after the peeling and the introduction of asample or the like, the importance of this property related to theattaching and peeling can be lessened.

An explanation will be given next regarding a method of using the PCRreaction vessel 210 configured as described above. First, a sample to beamplified through a thermal cycle is prepared. The sample includes thoseobtained by adding a plurality of types of primers, a thermostableenzyme and four types of deoxyribonucleoside triphosphates (dATP, dCTP,dGTP, dTTP) as PCR reagents to a mixture containing two or more types ofDNA. Next, the third sealing film 222 is peeled off from the substrate214 such that the first sample introduction port 233 and the secondsample introduction port 234 are open.

The sample is then introduced to either one of the first sampleintroduction port 233 and the second sample introduction port 234 by adropper, a syringe, or the like. FIG. 18 schematically shows a conditionwhere a sample 270 is introduced into the PCR reaction vessel 210. InFIG. 18 , in order to emphasize the position of the sample 270, thesample 270 is shown by a solid line that is thicker than that for thechannel 212. It should be noted that the solid line does not indicate astate where the sample 270 overflows outside the channel.

As shown in FIG. 18 , the sample 270 introduced to either one of thefirst sample introduction port 233 and the second sample introductionport 234 fills the channel by being pushed in by a dropper, a syringe,or the like, or by a capillary phenomenon. The sample 270 is packed inthe buffer channel region 212 f located between the first branch point212 c and the second branch point 212 d in the channel 212. However, thesample 270 does not enter the side of the thermal cycle region 212 e orthe second air communication port 26 of the channel 212 beyond the firstbranch point 212 c and the second branch point 212 d, which arerespectively located at both ends in the buffer channel region 212 f.This is because the both ends of the channel (i.e., the first aircommunication port 224 and the second air communication port 226) aresealed at this point such that there is no outlet for the air to escape.

Next, as shown in FIG. 19 , the third sealing film 222 is attached backonto the substrate 214 again so as to seal the first sample introductionport 233 and the second sample introduction port 234. As describedabove, a new third sealing film 222 may be attached. This completes theintroduction of the sample 270 into the PCR reaction vessel 210.

FIG. 20 is a diagram for explaining a PCR device 300 in which the PCRreaction vessel 210 is used. FIG. 21 is a diagram for explaining a statewhere the PCR reaction vessel 210 is set at a predetermined position ofthe PCR device 300.

The PCR device 300 is provided with a fluorescence detection opticalprobe 2122, a first heater 2134, and a second heater 2135. As shown inFIG. 21 , in the PCR reaction vessel 210, a pair of reaction regions ofthe thermal cycle region 212 e of the channel 212 is arranged on thefirst heater 2134 and the second heater 2135, respectively, and thefluorescence detection optical probe 2122 is installed in the PCR device300 so as to be located in a connection region between the pair ofreaction regions. For the PCR device 300, the PCR device applied in thePCR reaction vessel according to the first embodiment can be employed.

The PCR device 300 is further provided with a pump system 2110 forcausing the sample 270 to reciprocate in the thermal cycle region 212 e.This pump system 2110 is provided with a first nozzle 2101, a secondnozzle 2102, a first pump 2103, a second pump 2104, a first driver 2105,a second driver 2106, and a control unit 2107. The first nozzle 2101 ofthe pump system 2110 is connected to the first air communication port224 of the PCR reaction vessel 210, and the second nozzle 2102 of thePCR reaction vessel 210 is connected to the second air communicationport 226 of the PCR reaction vessel 210. A specific method forconnecting the nozzles to the respective air communication ports will bedescribed later. The pump system 2110 moves the sample in the thermalcycle region 212 e by controlling the pressure inside the channel 212via the first air communication port 224 and the second aircommunication port 226.

In the PCR device 300 according to the second embodiment, the firstheater 2134 and the second heater 2135 are set to differenttemperatures. Each heater provides the amount of heat to individuallycontrol the temperatures of a pair of reaction regions in the thermalcycle region 212 e and may be a means or a structure such as resistiveheating or a Peltier element. For example, the first heater 2134 iscontrolled by the first heater driver 2130 so as to maintain thetemperature of the reaction region on the right side of the figure pagein the thermal cycle region 212 e of the channel 212 to be 94° C.constantly. Also, the second heater 2135 is controlled by the secondheater driver 2132 so as to maintain the temperature of the reactionregion on the left side of the figure page to be 60° C. constantly. Thetemperature of each reaction region may be measured by a temperaturesensor (not shown) such as a thermocouple, and the output to each heatermay be controlled by each driver based on an electric signal therefrom.In this manner, the first heater 2134, the second heater 2135, the firstheater driver 2130, the second heater driver 2132, and the temperaturesensor may constitute a temperature adjustment unit for adjusting thetemperature of the thermal cycle region 212 e and may include otherelements that improve the controllability of the temperature. Thistemperature adjustment unit allows the thermal cycle region 212 e of thechannel 212 to be divided into two region of different atmospherictemperatures. Near each heater, a temperature sensor (not shown) such asa thermocouple that measures the temperature of a corresponding part maybe included, and other structures that improve the controllability ofthe temperature may be included. In the following, the reaction regionat the atmospheric temperature of 94° C. in the channel 212 is referredto as “high temperature part 2111”, and the reaction region at theatmospheric temperature of 60° C. in the channel 212 is referred to as“medium temperature part 2112”. Further, in the present embodiment, adetailed explanation will be given regarding a PCR device that isprovided with a PCR reaction vessel provided with a thermal cycle regionwhere temperature ranges of two levels are set as two reaction regionsand that is provided with a temperature control unit. However, the PCRdevice may be provided with a PCR reaction vessel provided with athermal cycle region where temperature ranges of three or more levelscan be set and that is provided with a temperature control unit. In thiscase, (although not shown), for example, the PCR device may be providedwith a PCR reaction vessel provided with reaction regions in which a lowtemperature part, a medium temperature part, and a high temperature partare arranged from the left side of the figure page and with atemperature control unit. In such a case, for example, the lowtemperature part, the medium temperature part, and the high temperaturepart are controlled to maintain 50 to 70° C., at 72° C., and 94° C.,respectively.

The pump system 2110 is arranged to cause the sample 270 to reciprocatewithin the thermal cycle region 212 e of the channel 212, as describedabove. By alternately operating the first pump 2103 and the second pump2104 through the first driver 2105 and the second driver 2106 under acertain condition by the control unit 2107, the sample 270 can bereciprocated between the high temperature part 2111 and the mediumtemperature part 112 of the channel 212, and a thermal cycle can beapplied to the sample 270 under a certain condition. In the PCR device300 according to the second embodiment, the first pump 2103 and thesecond pump 2104 are air pumps or blower pumps of a type where, whenboth the first pump 2103 and the second pump 2104 are stopped, theatmospheric pressures on a primary side and a secondary sideinstantaneously become equal to each other, and when both first pump2103 and the second pump 2104 are being stopped, the atmosphericpressure on the primary side and the atmospheric pressure on thesecondary side are equal to each other. If this type of pump is notused, that is, if a pump is used that maintains the immediatelypreceding pressure even when stopped, there is a possibility that aphenomenon occurs where the sample continues to move slightly even inthe case where the pump is stopped such that the sample does not stop ina predetermined reaction region and the temperature of the sample cannotbe appropriately controlled. On the other hand, external air and thechannel of the PCR reaction vessel communicate with each other in termsof the atmospheric pressure when stopped (when opened), having equalatmospheric pressure; however, since a filter is provided between theair communication port and the channel, contamination into the channelcan be prevented.

The sample 270 can undergo PCR by the above-described thermal cycle, andthe fluorescence from the sample 270 in the channel can be detected, andthe value thereof can be used as an index serving as information fordetermining the progress of the PCR or the termination of the reaction.As the fluorescence detection optical probe 2122 and the driver 2121,optical fiber-type fluorescence detector FLE-510 (manufactured by NipponSheet Glass Co., Ltd.) can be used, which is a very compact opticalsystem that allows for rapid measurement and the detection offluorescence regardless of a light and/or dark atmosphere. This opticalfiber type fluorescence detector can be also arranged easily in a narrowspace between the two temperature regions in the thermal cycle region.This optical fiber type fluorescence detector allows the wavelengthcharacteristic of the excitation light/fluorescence to be tuned suchthat the wavelength characteristic is suitable for the fluorescencecharacteristic of the sample 270 and thus allows an optimum optical anddetection system for a sample having various characteristics to beprovided. Further, fluorescence detection optical probes 2122 anddrivers 2121 may be provided that are installed at a plurality of sitesthroughout the thermal cycle region 212 e. For example, the fluorescencedetection optical probes 2122 and drivers 2121 may be installed todetect the fluorescence from the sample 270 in the channel located inthe high temperature part 111 or the medium temperature part 2112. Inaddition to the function of acquiring information for determining theprogress or the termination of the PCR, the fluorescence detectionoptical probes 2122 and drivers 2121 can also function as positionsensors for detecting, without fail, whether or not the sample 270 is inthe high temperature part 2111 or the medium temperature part 2112.

In the PCR device 300 configured as described above, the control unit2107 of the pump system 2110, the driver 2121 of the fluorescencedetection optical probe 2122, the first heater driver 2130, and thesecond heater driver 2132 are controlled to operate optimally by a CPU2141. Also, as described above, in the case where a reaction region inwhich temperatures od three levels are set, a third heater driver (notshown) is also controlled by the CPU in addition to the above.

FIG. 22 is a diagram showing a condition where a nozzle of a pump systemand an air communication port of the PCR reaction vessel are connected.FIG. 23 is a cross-sectional view of the PCR reaction vessel 210 shownin FIG. 23 that is sectioned along line C-C. As described above, thefirst nozzle 2101 is connected to the first air communication port 224,and the second nozzle 2102 is connected to the second air communicationport 226.

As shown in FIG. 23 , a needle 2150 is provided at the tip of the firstnozzle 2101. By perforating the first sealing film 218 with this needle2150, the first nozzle 2101 is connected to the first air communicationport 224. The same applies to the connection between the second nozzle2102 and the second air communication port 226.

The needle 2150 is provided with a packing material 2151 made of a softresin that comes into close contact with the surface of a sealing filmin order to secure airtightness around the connection. Immediately afterthe PCR reaction vessel 210 is set in the PCR device 300, the pumpsystem 2110 is not in operation and is open to the atmospheric air, andthe pressure inside the channel is thus in a state where the pressure isequal to the atmospheric pressure.

FIG. 24 shows a condition where the pump system 2110 is operated so asto move the sample 270. Either one of the first pump 2103 and the secondpump 2104 is operated so as to move the sample 270 from the bufferchannel region 212 f of the channel 212 to the high temperature part2111 or the medium temperature part 2112 of the thermal cycle region 12e. In FIG. 24 , the second pump 2104 to which the second nozzle 2102 isconnected is operated, and the first pump 2103 to which the first nozzle2101 is connected is stopped. In other words, the first aircommunication port 224 to which the first nozzle 2101 extending from thefirst pump 2103 is connected is open to the atmospheric pressure. Whenthe second pump 2104 is operated to feed the air from the second nozzle2102 to the second air communication port 226, the sample 270 moves fromthe buffer channel region 212 f of the channel 212, passes through themedium temperature part 2112, and moves to the high temperature part2111. This state is assumed to be an initial state.

More specifically, at the start of the operation of the second pump 2104or immediately before the start of the operation of the second pump2104, monitoring of the fluorescence emitted from the inside of thechannel is started using the fluorescence detection optical probe 2122.When there is nothing at a measurement point of the fluorescencedetection optical probe 2122, fluorescence that is detected is at zeroor at a background level. When the sample 270 exists at the measurementpoint, fluorescence is detected. Therefore, monitoring of thefluorescence is started at the start of the operation of the second pump2104, the completion of the movement of the sample 270 to the hightemperature part 2111 is recognized through a fluorescence value risingfrom the background level and dropping to the background level again,and stopping the operation of the second pump 2104 at this pointcompletes the setting of the initial state. Also, when the fluorescencedetection optical probe 2122 is further located in the high temperaturepart 2111, it is also possible to stop the sample 270 at the hightemperature part 2111 more certainly.

It should be noted that the sample 270 located inside the first branchedchannel 231 and the second branched channel 232 stays at the place evenwhen the second pump 2104 is operated. This is because the first sampleintroduction port 233 and the second sample introduction port 234 aresealed with the third sealing film 222. The sample 270 located insidethe first branched channel 231 and the second branched channel 232 isnot subjected to PCR. Therefore, even when the amount of a sample thatis initially introduced to the PCR reaction vessel 210 varies, bysetting the volume of the buffer channel region 212 f of the channel 212formed in the PCR reaction vessel 210 to a predetermined volumeaccording to the amount of the sample on which a PCR process is intendedto be performed, a certain desired amount of sample can be always sentto the thermal cycle region 212 e of the channel 212, and a fluorescenceamount that affects the determination on the progress or the terminationof PCR can be kept approximately constant. In other words, the bufferchannel region 212 f of the channel 212 has a dispensing function thatallows for the extraction of a certain desired amount of sample.

After the setting to the initial state, a thermal cycle is applied tothe sample 270 to progress the PCR. The measurement of fluorescence bythe fluorescence detection optical probe 2122 is continued.

(A) First, the sample 270 is allowed to sit for 1 to 30 seconds in thehigh temperature part 2111 (about 94° C. atmosphere) (denaturation:thermal denaturation step). Through this step, double-stranded DNA isdenatured into single strands.

(B) Next, the first pump 2103 to which the first nozzle 2101 isconnected is operated to move the sample 270 to the medium temperaturepart 2112 (about 60° C. atmosphere). More specifically, the sample 270is pushed in a direction from the high temperature part 2111 to themedium temperature part 2112 by the action of the first pump 2103. Sincethe fluorescence measurement by the fluorescence detection optical probe2122 continues, the operation of the first pump 2103 is stopped at thepoint of time when a fluorescence amount rises from the background leveland drops again due to the sample 270 passing through the measurementpoint of the fluorescence detection optical probe 2122 (or after acertain period of time has passed after the fluorescence amount hasdecreased). Further, when the fluorescence detection optical probe 2122is located at the medium temperature part 2112, it is also possible tomore certainly stop the sample 270 at the medium temperature part 2112.

(C) In the medium temperature part 2112, the sample 270 is allowed tosit for 3 to 60 seconds (annealing+elongation: annealing step+elongationstep). Through these steps, binding of primers contained in the sample270 in advance occurs resulting in further elongated DNA.

(D) Next, the second pump 2104 to which the second nozzle 2102 isconnected is operated to move the sample 270 from the medium temperaturepart 2112 to the high temperature part 2111. The timing for stopping thepump operation is determined based on changes in the fluorescence amountmeasured by the fluorescence detection optical probe 2122 in the samemanner as described above. After moving the sample 270 to the hightemperature part 2111, the sample 270 is allowed to sit for 1 to 30seconds to go through heat denaturation.

(E) By repeating the above (B) to (D) for a predetermined number ofcycles, applying a thermal cycle to the sample 270, and allowing the DNAcontained in the sample 270 to undergo a plurality of cycles of thermaldenaturation, annealing, and elongation steps, the amplification of DNAis performed. The number of cycles is appropriately determined by acombination of target DNA, primers, enzymes, and the like.

After the completion of a predetermined number of thermal cycles, thefirst pump 2103 and the second pump 2104 are stopped, and the PCR isended. Even when the predetermined number of thermal cycles are applied,the fluorescence is measured by the fluorescence detection optical probe122, and the fluorescence detected from the sample 270 increases as theDNA contained in the sample 270 is amplified. Thereby, the concentrationof the sample 270 can be accurately known.

According to the PCR reaction vessel 210 according to the secondembodiment, by providing the first filter 228 between the first aircommunication port 224 and the channel 212 and the second filter 230between the second air communication port 226 and the channel 212,contamination inside the channel 212 can be prevented. Although theimplementation of measures to prevent contamination on the side of thepump system 2110 is likely to increase the cost, in the PCR reactionvessel 210 according to the second embodiment, the measures allowcontamination to be prevented only on the PCR reaction vessel 210 sideand are thus economical. Further, when the PCR reaction vessel is usedas a disposable vessel, since the filter is always a new one,contamination can be further prevented at low cost. Furthermore,regarding the disposal of the PCR reaction vessel, since the sample issubstantially sealed in the PCR reaction vessel, the disposal is alsomeaningful in terms of safety and environment.

Further, according to the PCR reaction vessel 210 according to thesecond embodiment, by providing the buffer channel region in the channel212, a sample subjected to PCR can be dispensed, and only a requiredamount of sample can be always sent to the thermal cycle region of thechannel 212.

In the PCR device 300 according to the second embodiment, the sample canbe caused to reciprocate inside the channel 212 of the PCR reactionvessel 210 by alternately operating the first pump 2103 and the secondpump 2104 that allow the pressures on the primary side and the secondaryside to become equal when stopped. In this case, since excessivepressure is not applied to the sample during the liquid feeding(applying pressure to the sample in the channel) and, further, thepressure in the channel is not reduced, evaporation and boiling(foaming) of the liquid containing the sample due to the action of thehigh temperature part 2111 can be prevented.

Further, in the PCR device 300 according to the second embodiment, thefluorescence from the sample is monitored all the time even during PCRin the thermal cycle region (real-time PCR). As a result, the end timingof the PCR can be determined based on the fluorescence amount that hasbeen measured. Further, by monitoring a change in fluorescence by thefluorescence detection optical probe 2122, the passing of the sample canbe known, and, based on the change in the fluorescence amountaccompanying the passing of the sample, the alternate operation of thefirst pump 2103 and the second pump 2104 can be controlled. Thus, thesample to be subjected to the PCR can be accurately positioned to thehigh temperature part 2111 or the medium temperature part 2112 of thethermal cycle region.

On the other hand, in the case of a PCR reaction vessel and a PCR devicehaving a reaction region where the above-described temperatures of threelevels: the high temperature part; the medium temperature part; and thelow temperature part, are controlled, the steps, heat denaturation,annealing, and elongation, can be performed at the high temperaturepart, at the medium temperature part, and at the low temperature part,respectively. Also, the control thereof can be easily developed andimproved by those skilled in the art based on the above detaileddescription. Those skilled in the art can appropriately choose, based onthe characteristics of the sample, whether the reaction region is set tohave two levels or three levels.

Described above is an explanation of the present invention based on theembodiments. These embodiments are intended to be illustrative only, andit will be obvious to those skilled in the art that variousmodifications to constituting elements and processes could be developedand that such modifications are also within the scope of the presentinvention.

In the above-stated embodiments, a pair of pumps that allows pressureson a primary side and a secondary side to be equal when stopped isarranged at the respective ends of a channel. Alternatively, a pumpcapable of pressurization and suctioning may be provided only at eitherone of the ends of the channel, and the other end may be open to theatmospheric pressure. In other words, a sample is moved in a thermalcycle region by controlling the pressure inside a channel via a firstair communication port and a second air communication port. In thiscase, a process of switching the operations of a pair of pumps at afixed timing is not necessary, and pump controlling is thus facilitated.

Also, in the above-described embodiments, a measurement point of afluorescence detection optical probe is arranged between a hightemperature part and a medium temperature part. Alternatively, ameasurement point of a fluorescence detection optical probe may bearranged at each of the high temperature part and the medium temperaturepart. In this case, the accuracy for positioning a sample can beincreased.

What is claimed is:
 1. A PCR reaction vessel comprising: a substrate; achannel formed on the substrate; a pair of filters that is provided atrespective ends of the channel and that is for preventing contaminationinside the channel; a pair of air communication ports that communicatewith the channel through the respective filters; a sample introductionport for introducing a sample that is subjected to PCR inside thechannel; and a thermal cycle region that is formed between the pair offilters in the channel and that includes a plurality of differenttemperature regions capable of causing PCR in the sample; wherein thesample repeatedly moves in a reciprocating manner between the pluralityof temperature regions, wherein the substrate is a parallel plate-shapedsubstrate and has a first surface and a second surface that faces thefirst surface, wherein the channel is formed on the first surface of thesubstrate, wherein the air communication ports are formed on the secondsurface of the substrate, wherein the thickness of the filters issmaller than the thickness of the substrate, wherein the filters fit ina filter installation space formed at an intermediate position betweenthe first surface and the second surface in the substrate, and whereinair sent to the air communication ports passes through the filters fromthe second surface, reaches the first surface, and is introduced intothe channel.
 2. The PCR reaction vessel according to claim 1, whereinthe plurality of temperature regions each include a serpentine channel.3. The PCR reaction vessel according to claim 1, further comprising: asealing film for sealing the air communication ports, the filters, andthe sample introduction port; and a channel sealing film for sealing atleast the plurality of temperature regions in the channel.
 4. The PCRreaction vessel according to claim 1, wherein the thermal cycle regionincludes a high temperature part for causing heat denaturation in thesample and a medium temperature part for causing annealing andelongation.
 5. The PCR reaction vessel according to claim 4, wherein thedistance between the sample introduction port and the medium temperaturepart is smaller than the distance between the sample introduction portand the high temperature part.
 6. A PCR device comprising: the PCRreaction vessel according to claim 1; a temperature adjustment unit foradjusting the temperature of the thermal cycle region; and a pump systemthat is connected to the air communication ports and controls thepressure inside the channel in order to move the sample inside thethermal cycle region.
 7. The PCR device according to claim 6, whereinthe pump system is provided with a pair of pumps respectively connectedto the pair of air communication ports, wherein pressure on the primaryside and pressure on the secondary side become equal in the pumps whenthe pumps are stopped, and wherein, by alternately operating the pair ofpumps, the sample is caused to repeatedly move in a reciprocating mannerbetween the plurality of temperature regions.
 8. The PCR deviceaccording to claim 6, wherein the pump system is provided with a hollowneedle, and wherein perforation of the sealing film for sealing the aircommunication ports with the needle causes the pump system and thechannel to communicate with each other.
 9. The PCR device according toclaim 6, further comprising a fluorescence detector for detectingfluorescence generated from the sample inside the channel.
 10. The PCRdevice according to claim 9, wherein the thermal cycle region isprovided with a connection region connecting the plurality oftemperature regions, and wherein the fluorescence detector detectsfluorescence from the connection region.
 11. The PCR device according toclaim 9, further comprising a control unit for controlling the pumpsystem based on a value detected by the fluorescence detector, whereinthe control unit determines the progress of PCR based on the valuedetected by the fluorescence detector.
 12. A PCR method comprising:preparing a PCR reaction vessel including: a parallel plate-shapedsubstrate; a channel formed on the substrate; a pair of filters that fitin installation spaces formed at respective ends of the channel andwhose thickness is smaller than the thickness of the substrate; a pairof air communication ports that communicate with the channel through therespective filters; a sample introduction port for introducing a samplethat is subjected to PCR inside the channel; and a thermal cycle regionthat is formed between the pair of filters in the channel and thatincludes a plurality of different temperature regions capable of causingPCR in the sample; controlling the respective temperatures of theplurality of temperature regions in the thermal cycle region such thatPCR of the sample is possible; connecting a pump system for controllingthe pressure inside the channel to the air communication ports; andcontrolling the pressure applied to the sample inside the channel andrepeatedly moving the sample in a reciprocating manner between theplurality of temperature regions in the thermal cycle region byoperating the pump system so as to cause PCR in the sample, wherein thesubstrate has a first surface and a second surface that faces the firstsurface, wherein the channel is formed on the first surface of thesubstrate, wherein the air communication ports are formed on the secondsurface of the substrate; wherein the installation spaces are formed atan intermediate position between the first surface and the secondsurface in the substrate, and wherein air sent to the air communicationports passes through the filters from the second surface, reaches thefirst surface, and is introduced into the channel.
 13. The PCR methodaccording to claim 12, wherein the thermal cycle region includes a hightemperature part for causing heat denaturation in the sample and amedium temperature part for causing annealing and elongation.
 14. ThePCR method according to claim 12, wherein the thermal cycle regionincludes a high temperature part for causing heat denaturation in thesample, a medium temperature part for causing annealing, and a lowtemperature part for causing elongation.
 15. The PCR method according toclaim 12, wherein the PCR reaction vessel further includes: a sealingfilm for sealing the air communication ports, the filters, and thesample introduction port; and a channel sealing film for sealing atleast the plurality of temperature regions in the channel.
 16. The PCRmethod according to claim 12, wherein the pump system is provided with ahollow needle, further comprising: perforating the sealing film forsealing the air communication ports with the needle so as to cause thepump system and the channel to communicate with each other.
 17. The PCRmethod according to claim 12, wherein the thermal cycle region isprovided with a connection region connecting the plurality oftemperature regions, further comprising: detecting fluorescencegenerated from the sample inside the connection region; and monitoringthe detected fluorescence.
 18. The PCR method according to claim 17,further comprising controlling the pump system based on a change in thedetected fluorescence.